Bottom Line:
We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes.ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks.Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading.

gkt494-F5: Effects of topo IIα depletion and addition on DNA replication kinetics and nuclear structure. (A and B) Topo IIα immunodepletion. Mock-depleted and topo IIα-depleted extracts were analyzed by western blotting with an anti-Xtopo IIα antibody (A; volume of extracts are indicated) and by kDNA decatenation (B). (C) Sperm nuclei were incubated in mock-depleted or topoIIα-depleted extracts containing [α-32P]-dATP plus or minus 100 µM ICRF-193 for 60 min, and the ratio of replicated DNA with and without ICRF-193 was calculated. (D) Sperm nuclei were incubated in mock-depleted (dotted lines) or topo IIα-depleted (solid lines) extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares), and replication efficiency was measured at the indicated times. (E) Sperm nuclei were incubated in mock-depleted (upper panels) or topo IIα-depleted (lower panels) extracts in the presence of rhodamin–dUTP for 90 min, fixed and stained with Hoechst and observed under a fluorescence microscope. Scale bar, 2.5 µm. (F) Sperm nuclei were incubated in extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares) added at 0 min, and replication was measured at the indicated times. (G) Same as in (F), except that buffer or topo IIα were added at 20 min.

Mentions:
The prereplicative effects of ICRF-193 could result from catalytic inhibition of topo IIα or from trapping topo IIα clamps on chromatin or perhaps from some other mechanism unrelated to topo IIα inhibition. To address these questions, we examined the effects of topo IIα immunodepletion on sperm chromatin decondensation and replication. The extent of topo IIα immunodepletion was estimated to >95% by western blot (Figure 5A) and >99% by kDNA decatenation (Figure 5B). Addition of ICRF-193 to mock-depleted extracts inhibited DNA replication as with undepleted extracts. However, topo IIα depletion completely abolished the effect of ICRF-193 on DNA replication as judged by [α-32P]dATP incorporation (Figure 5C). This result shows that ICRF-193 has no detectable effect on DNA replication other than by topo IIα inhibition.Figure 5.

gkt494-F5: Effects of topo IIα depletion and addition on DNA replication kinetics and nuclear structure. (A and B) Topo IIα immunodepletion. Mock-depleted and topo IIα-depleted extracts were analyzed by western blotting with an anti-Xtopo IIα antibody (A; volume of extracts are indicated) and by kDNA decatenation (B). (C) Sperm nuclei were incubated in mock-depleted or topoIIα-depleted extracts containing [α-32P]-dATP plus or minus 100 µM ICRF-193 for 60 min, and the ratio of replicated DNA with and without ICRF-193 was calculated. (D) Sperm nuclei were incubated in mock-depleted (dotted lines) or topo IIα-depleted (solid lines) extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares), and replication efficiency was measured at the indicated times. (E) Sperm nuclei were incubated in mock-depleted (upper panels) or topo IIα-depleted (lower panels) extracts in the presence of rhodamin–dUTP for 90 min, fixed and stained with Hoechst and observed under a fluorescence microscope. Scale bar, 2.5 µm. (F) Sperm nuclei were incubated in extracts containing [α-32P]-dATP supplemented with buffer (circles) or with recombinant human topo IIα (squares) added at 0 min, and replication was measured at the indicated times. (G) Same as in (F), except that buffer or topo IIα were added at 20 min.

Mentions:
The prereplicative effects of ICRF-193 could result from catalytic inhibition of topo IIα or from trapping topo IIα clamps on chromatin or perhaps from some other mechanism unrelated to topo IIα inhibition. To address these questions, we examined the effects of topo IIα immunodepletion on sperm chromatin decondensation and replication. The extent of topo IIα immunodepletion was estimated to >95% by western blot (Figure 5A) and >99% by kDNA decatenation (Figure 5B). Addition of ICRF-193 to mock-depleted extracts inhibited DNA replication as with undepleted extracts. However, topo IIα depletion completely abolished the effect of ICRF-193 on DNA replication as judged by [α-32P]dATP incorporation (Figure 5C). This result shows that ICRF-193 has no detectable effect on DNA replication other than by topo IIα inhibition.Figure 5.

Bottom Line:
We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes.ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks.Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading.